Application of beta-sitosterol in preparation of medicine for treating inflammatory diseases

Abstract

The invention discloses application of beta-sitosterol in preparation of a medicine for treating inflammatory diseases, and belongs to the field of biological medicine. The medicine is a gene expression inhibitor, the beta-sitosterol realizes an anti-inflammatory effect by inhibiting expression of inflammasome related genes Drkat7b, Drnek7 and Drcaspase-1, particularly, the medicine takes the beta-sitosterol or a pharmaceutically acceptable solvate thereof as an active ingredient, the use concentration is 0.1-100 mu M, and the dosage of the beta-sitosterol or the pharmaceutically acceptable solvate of the beta-sitosterol or the pharmaceutically acceptable solvate of the beta-sitosterol or the pharmaceutically acceptable solvate of the beta-sitosterol or the pharmaceutically acceptable solvate of the beta-sitosterol is 0.1-100 mu M. The invention also provides application of beta-sitosterol in preparation of drugs for treating inflammatory diseases induced by CuSO4, and / or LPS, and / or DSS. According to the invention, a model for inhibiting zebrafish juvenile fish inflammation induced by CuSO4, LPS and DSS by beta-sitosterol is constructed, and the beta-sitosterol is verified to inhibit inflammation caused by different irritants by inhibiting expression of zebrafish inflammasome related genes, so that an anti-inflammatory effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of β-sitosterol in the preparation of drugs for treating inflammatory diseases. Background Art

[0002] Inflammatory response is a physiological response triggered by the innate immune system of the body. When the body detects various harmful stimuli, it will activate a self-protective immune response, thus forming an inflammatory response. The immune system is the first line of defense for the body against pathogenic microorganisms or harmful stimuli. Pattern recognition receptors (PRRs) in the body can respond to various external stimuli, thereby triggering an immune response, and this immune response will activate downstream related inflammatory pathways, inducing an inflammatory response. By controlling the inflammatory response, the body can repair damaged tissues or eliminate microbial infections. However, excessive inflammatory response will lead to the occurrence of chronic persistent or systemic inflammatory diseases, which is an important factor leading to the occurrence and aggravation of many diseases. A large part of the body's inflammatory response is triggered by pathogenic microorganisms and the molecules they transport or release. These molecules are called pathogen-associated molecular patterns (PAMPs), including bacterial flagellin, lipopolysaccharide (LPS), peptidoglycan, and viral double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA), etc. The innate immune system of the body will be activated by PAMPs, resulting in the infiltration of inflammatory cells and the release of cytokines. All of these will cause long-term inflammatory tissue damage, which is an important pathological basis for many chronic diseases. In recent years, it has been found that tissues or cells will release endogenous molecules with immunomodulatory effects when they are damaged, infected or stressed. These molecules enter the intercellular space or blood and are recognized by PRRs, which will trigger an aseptic inflammatory response, leading to the occurrence and development of certain inflammatory diseases. Different from PAMPs, these endogenous molecules are called damage-associated molecular patterns (DAMPs).

[0003] The inflammasome is a multimeric complex composed of cytoplasmic proteins that responds to DAMPs and PAMPs and plays an important role in the occurrence and development of inflammation-related diseases and innate immunity by mediating inflammatory responses. The inflammasome consists of three parts: the pattern recognition receptor sensor (PRR), the adaptor, and the effector molecule pro-caspase-1. Multiple inflammasomes have been discovered, including NLRP1, NLRP2, NLRP3, NLRP4, NLRP6, NLRP7, NLRP12, AIM2, and NAIP, etc. Among them, the NLRP3 inflammasome is the most studied and best understood type. The NLRP3 inflammasome is composed of a sensor (NLRP3), an adaptor (ASC, also known as PYDARD), and an effector Caspase-1. Under normal circumstances, the NLRP3 inflammasome is in an inactive or inhibited state, but it will be activated when the body detects the invasion of pathogens or other alarm signals. The activation process of the NLRP3 inflammasome includes two steps. One is the priming step, which promotes the expression of inflammasome components; the other is the assembly step, which needs to be triggered by several specific molecules that activate NLRP3.

[0004] The hallmark responses of inflammasome priming include the activation or proteolytic cleavage of Caspase-1, the secretion of bioactive cytokines IL-1β and IL-18, and the induction of pyroptosis. Priming is activated by different extracellular signals, such as PAMPs (e.g., TLR ligands or NOD2 ligands) or cytokines (TNF-α, IL-1β, IFN-I). In addition, DAMPs or alarmins detected by various receptors, such as double-stranded DNA, mitochondrial DNA, ATP, reactive oxygen species (ROS), heme, or uric acid, can also activate immune cells in a non-infectious environment, especially through the activation of the NF-κB pathway.

[0005] NIMA-related kinase 7 (NEK7) belongs to the serine / threonine kinase family. NEK7 is a multifunctional kinase that can affect centrosome replication, mitochondrial regulation, intracellular protein trafficking, DNA repair, and spindle assembly. Recent studies have shown that NEK7 is related to the LRR domain of NLRP3, and it binds to the LRR domain of NLRP3 in a kinase-independent role, promoting the formation of the NLRP3-ASC complex, ASC oligomerization, and Caspase-1 activation, thereby activating the NLRP3 inflammasome. In addition, NF-κB has been shown to upregulate the expression of NLRP3, and the subunit RELA of NF-κB can regulate the transcription of NEK7, and thus regulate the expression of NEK7. When these two interactions occur simultaneously, the interaction between NEK7 and NLRP3 may increase, further supporting the important role of NEK7 in NLRP3 activation. Other studies have confirmed that K + efflux is an important factor in activating the NLRP3 inflammasome, and NEK7 is a specific K + sensor that can enhance the interaction between NEK7 and NLRP3 when homeostasis is disturbed and intracellular K + efflux increases, thereby activating the NLRP3 inflammasome.

[0006] The acetylation state is considered a key factor in regulating the transcription of inflammatory genes. For example, MAPK phosphatase 1 (MKP1) is a dual-specific phosphatase that can be acetylated by TLR agonists. At this time, the interaction between MKP1 and p38 is enhanced, resulting in a decrease in the phosphorylation of p38, thereby negatively regulating the TLR-induced inflammatory response. Histone acetyltransferases and histone deacetylases jointly regulate the acetylation state of histones. The MYST family, which belongs to one of the histone acetyltransferase (HAT) protein families, is one of the largest known acetyltransferase families. Members of this family are widely expressed in cells and all contain a highly conserved lysine acetyltransferase domain, with important biological functions. The mammalian MYST family consists of five members: KAT5 (Htatip / Tip60), KAT6A (MYST3 / MOZ), KAT6B (MYST4 / MORF), KAT7 (MYST2 / HBO1), and KAT8 (MYST1 / MOF). Zhao et al. found that during the complete activation of the NLRP3 inflammasome, acetylation of NLRP3 occurs, which can accelerate the polymerization of NLRP3 and its interaction with ASC and NEK7, thereby promoting the assembly of the inflammasome. Zhang et al. further confirmed through the construction of various transgenic mice, molecular biochemical experiments, and live cell real-time imaging that KAT5 mediates the acetylation of NLRP3, and this acetylation modification is a key event for NLRP3 oligomerization and activation.

[0007] Lysine acetyltransferase 7 (KAT7) is a typical member of the MYST family. KAT7 is also commonly referred to as HBO1, HBOA, MYST2, or ZC2HC7. It mainly acetylates histone H3 and H4, thereby regulating gene replication or transcription. KAT7 has multiple functions. In addition to participating in histone acetylation, it can also propionylate and ubiquitinate proteins. Moreover, KAT7 also has functions such as promoting tissue-specific gene expression, promoting T cell development, and regulating cell senescence. Under normal circumstances, KAT7 mediates histone acetylation, enabling transcription factors to bind to chromatin and regulate transcription initiation, or the KAT7 complex occupies the coding region and plays a direct role in transcription elongation. KAT7 can also be used to acetylate replication factors, thereby changing the interactions between proteins in the DNA replication initiation complex, thus affecting DNA replication.

[0008] Zebrafish (Danio rerio) is an ornamental fish native to the south subtropical region and is widely used in scientific research fields such as biology and toxicology. It has become an ideal model for studying toxicology, diseases, immunity, and genetics. After the zebrafish genome was fully sequenced, it was found to be highly similar to the human genome, making it a new model organism. Compared with other model animals, zebrafish are easier to genetically manipulate. At the same time, zebrafish have a short reproductive cycle, a large number of eggs, stable species, insignificant individual differences, small size, and are convenient for breeding; zebrafish eggs are transparent and can develop in vitro, providing convenient conditions for embryo imaging; the organ development of zebrafish is very similar to that of mammals, and its immune system is highly conserved. The immune cells are similar in type and morphology to those of mammals, and the immune system is also divided into innate immunity and acquired immunity. Zebrafish respond rapidly to external stimuli, can be induced to produce oxidative stress and induce an inflammatory response, so zebrafish are also an ideal model for studying inflammatory responses and anti-inflammatory drugs. At the same time, zebrafish have transgenic lines with multiple immune cell fluorescence labels, providing convenient conditions for studying the migration and quantitative changes of immune cells.

[0009] The zebrafish LPS inflammation model is a systemic inflammation induced by lipopolysaccharide (LPS). LPS is an endotoxin in the cell wall of Gram-negative bacteria. The method for establishing the LPS inflammation model is to soak zebrafish embryos in an LPS solution or microinject LPS into the yolk sac, which can induce and stimulate zebrafish to produce more immune cells and trigger inflammation. Research has shown that zebrafish can respond to LPS stimulation. After LPS stimulates zebrafish, it induces the production of more NO and ROS in vivo and upregulates the mRNA expression levels of related pro-inflammatory cytokine genes. In this model, the indicator for measuring the degree of inflammation or the anti-inflammatory effect of drugs is the change in the number of immune cells in zebrafish. Studies using the LPS inflammation model have shown that the extract Polyphyllin VII (PP7) of Paris polyphylla can inhibit the production of NO, inhibit the heartbeat of model zebrafish, and reduce yolk sac edema; in the LPS model, treatment with chlorogenic acid can inhibit the migration of macrophages and neutrophils to the inflammation site; the extract of the leaves of Chimonanthus nitens can inhibit neutrophil recruitment and inhibit the expression of il6, il1β, and tnfα induced by LPS.

[0010] The CuSO4 inflammation model is an acute inflammation model induced by a CuSO4 solution. Copper is a trace element, but excessive copper ions in the environment can disrupt the homeostasis in zebrafish, thereby inducing an inflammatory response caused by oxidative stress. It has been found that CuSO4 can inhibit the survival and development of zebrafish embryos, cause the death of hair cells at the lateral line neuromasts of zebrafish, trigger a strong inflammatory response, and induce the migration of immune cells to this site. Therefore, CuSO4 exposure is often used to induce or simulate inflammation. The method for establishing the model is similar to that of the LPS inflammation model. Zebrafish can be directly soaked in the solution for induction, and combined with zebrafish lines with fluorescently labeled neutrophils for observation. The number of neutrophils at the neuromast is a commonly used indicator of the inflammation level in this model. There have been many studies on the effects of natural products using the zebrafish CuSO4 inflammation model, including natural products such as the root extract of Sanguisorba officinalis, the extract of Rutaceae Citrus changshan-huyou Y.B.Chang, PP7, and the leaf extract of Clerodendrum cyrtophyllum.

[0011] Dextran sulfate sodium (DSS) is a synthetic sulfated polysaccharide belonging to polyanionic derivatives. It is soluble in water and has good stability. It can affect the synthesis of intestinal wall cell DNA, inhibit epithelial cell proliferation, and damage the intestinal mucosal barrier, etc., and is quite similar to the inflammatory characteristics induced by trinitrobenzenesulfonic acid. Treating zebrafish larvae three days after fertilization with DSS will lead to excessive bacterial growth, infiltration of neutrophils into the intestine, upregulation of pro-inflammatory genes, and overall inhibition of cell proliferation, which is related to intestinal microbiota dysbiosis and DSS damage to the intestinal mucosal barrier. Different from the chronic murine inflammatory bowel disease (IBD) model induced by DSS which requires a modeling cycle of several months, in zebrafish, repetitive DSS injury significantly cannot fully recover, the mortality rate increases, mucus production is impaired, and autophagy is impaired, all of which occur within two weeks. Research has found that using the zebrafish IBD model induced by repetitive DSS to clarify that prostaglandin E2 plays a key role in protecting against invasion of intestinal barrier-related bacteria by increasing the production of mucin and blocking the uptake of luminal Escherichia coli proteins, and also proves that the drug mesalamine only works during intestinal injury and does not play a protective role during the recovery period after DSS removal. Some research has used the DSS-induced zebrafish IBD model to explore the positive effects of probiotics on the remission and prevention of IBD, as well as the effectiveness and safety of probiotics in treating IBD, established a method for rapidly screening probiotic candidate bacteria, and screened out a strain of bacteria with the best protective effect - Bacillus smithii XY1. There is also research using the DSS-induced zebrafish larval enteritis model to find that palmitoylethanolamide oxazoline can reduce DSS-induced intestinal injury and mucus production, and can reduce the expression of genes related to inflammation and endoplasmic reticulum stress.

[0012] β-Sitosterol is one of the most common plant sterols and is a major component of the cell membranes of animals and plants. β-Sitosterol belongs to 4-demethylsterols in classification and is the main component of 4-demethylsterols. It exists in a variety of fruits, vegetables and seeds, has a variety of pharmacological properties, its molecular weight is 414.7 Da, and its molecular formula is C 29 H 50 O, with a melting point of 140 °C and a boiling point of 501.9 °C (760 mmHg), and a density of 0.97 g / cm 3 . Under normal conditions, β-sitosterol is in a solid state, insoluble in water at room temperature, highly soluble in organic solvents but slightly soluble in ethanol, widely exists in plants, and has a relatively high content in vegetable oils, beans and seeds. β-Sitosterol is a natural product belonging to tetracyclic triterpenoids, and its chemical structure is similar to that of cholesterol. There is no relevant report in the prior art on β-sitosterol inhibiting inflammation induced by different stimulants such as CuSO4, LPS and DSS by inhibiting the expression of zebrafish inflammasome-related genes kat7b, nek7 and caspase-1. Summary of the Invention

[0013] In view of the above deficiencies in the prior art, the object of the present invention is to provide the use of β-sitosterol in the preparation of anti-inflammatory drugs. By constructing an inflammation model of zebrafish larvae induced by CuSO4, LPS and DSS, it is found that β-sitosterol can inhibit the inflammation induced by the above three different stimuli by inhibiting the expression of genes related to the inflammasome in zebrafish, thereby achieving an anti-inflammatory effect.

[0014] To achieve the above object, the present invention adopts the following technical solutions:

[0015] In the first aspect of the present invention, there is provided the use of β-sitosterol in the preparation of a drug for treating inflammatory diseases, wherein the drug is an inhibitor of the expression of genes related to the inflammasome, and the genes related to the inflammasome are selected from one or more of Drkat7b, Drnek7 and Drcaspase-1.

[0016] Preferably, the drug uses β-sitosterol or its pharmaceutically acceptable solvate as the active ingredient, and the use concentration is 0.1 - 100 μM.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable carrier or diluent.

[0018] Preferably, the dosage form of the drug is selected from at least one of tablets, capsules, granules, dripping pills, suspensions, syrups, enteric preparations, emulsion suspensions and injections.

[0019] In the second aspect of the present invention, there is provided a pharmaceutical composition for treating inflammatory diseases, comprising β-sitosterol or its pharmaceutically acceptable solvate as the active ingredient, and the use concentration is 0.1 - 100 μM.

[0020] In the third aspect of the present invention, there is provided the use of β-sitosterol in the preparation of a drug for treating inflammatory diseases induced by CuSO4, LPS and DSS.

[0021] Preferably, the drug is an inhibitor of the expression of genes related to the inflammasome, and the genes related to the inflammasome are selected from one or more of Drkat7b, Drnek7 and Drcaspase-1.

[0022] In the fourth aspect of the present invention, there is provided a pharmaceutical composition for treating inflammatory diseases induced by CuSO4, and / or LPS, and / or DSS, comprising β-sitosterol or its pharmaceutically acceptable solvate as the active ingredient, and the use concentration is 0.1 - 100 μM.

[0023] Compared with the prior art, the present invention constructs an inflammation model of zebrafish larvae induced by β-sitosterol against CuSO4, LPS, and DSS, and finds that β-sitosterol can inhibit inflammation caused by different stimulants such as CuSO4, LPS, and DSS by inhibiting the expression of genes related to the inflammasome in zebrafish, thereby achieving an anti-inflammatory effect. Brief Description of the Drawings

[0024] Figure 1 Changes in the expression of genes related to the inflammasome after administration following copper ion stimulation in the examples; (A) Changes in the expression of the Drkat7b gene; (B) Changes in the expression of the Drnek7 gene; (C) Changes in the expression of the Drcaspase-1 gene; compared with the group treated with CuSO4 alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0025] Figure 2 Changes in the expression of genes related to the inflammasome after copper ion stimulation following prior administration in the examples; (A) Changes in the expression of the Drkat7b gene; (B) Changes in the expression of the Drnek7 gene; (C) Changes in the expression of the Drcaspase-1 gene; compared with the group treated with CuSO4 alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0026] Figure 3 Changes in the expression of genes related to the inflammasome with simultaneous administration and copper ion stimulation in the examples; (A) Changes in the expression of the Drkat7b gene; (B) Changes in the expression of the Drnek7 gene; (C) Changes in the expression of the Drcaspase-1 gene; compared with the group treated with CuSO4 alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0027] Figure 4 Changes in the expression of genes related to the inflammasome after administration following LPS stimulation in the examples; (A) Changes in the expression of the Drkat7b gene; (B) Changes in the expression of the Drnek7 gene; (C) Changes in the expression of the Drcaspase-1 gene; compared with the group treated with LPS alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0028] Figure 5For the changes in the expression of inflammasome-related genes after administration followed by LPS stimulation in the examples; (A) Changes in the expression of Drkat7b gene; (B) Changes in the expression of Drnek7 gene; (C) Changes in the expression of Drcaspase-1 gene; compared with the group treated with LPS alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0029] Figure 6 For the changes in the expression of inflammasome-related genes with simultaneous administration and LPS stimulation in the examples; (A) Changes in the expression of Drkat7b gene; (B) Changes in the expression of Drnek7 gene; (C) Changes in the expression of Drcaspase-1 gene; compared with the group treated with LPS alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0030] Figure 7 For the changes in the expression of inflammasome-related genes after administration following DSS stimulation in the examples; (A) Changes in the expression of Drkat7b gene; (B) Changes in the expression of Drnek7 gene; (C) Changes in the expression of Drcaspase-1 gene; compared with the group treated with LPS alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0031] Figure 8 For the changes in the expression of inflammasome-related genes after administration followed by DSS stimulation in the examples; (A) Changes in the expression of Drkat7b gene; (B) Changes in the expression of Drnek7 gene; (C) Changes in the expression of Drcaspase-1 gene; compared with the group treated with LPS alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0032] Figure 9 For the changes in the expression of inflammasome-related genes with simultaneous administration and DSS stimulation in the examples; (A) Changes in the expression of Drkat7b gene; (B) Changes in the expression of Drnek7 gene; (C) Changes in the expression of Drcaspase-1 gene; compared with the group treated with LPS alone, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Detailed implementation manners

[0033] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all fall within the protection scope of the present invention.

[0034] Example 1

[0035] 1. Experimental materials and methods

[0036] 1.1 Zebrafish and experimental conditions

[0037] All zebrafish of the AB strain used in the experiment were purchased from the China Zebrafish Resource Center (CZRC). The laboratory carried out zebrafish breeding according to the breeding specifications of the reference website (zfin.org). Zebrafish were fed regularly 2 times a day. One night before spawning, male and female fish were placed in a mating fish tank at a ratio of 1:1; the next morning, the plate was removed to lay eggs 1 h after the fish house lights were turned on. The fertilized eggs were collected and placed in an incubator at 28°C, and the E3 culture medium was changed every 24 h. Zebrafish at 3 days post-fertilization were used for the experiment, and all zebrafish used were approved by the Experimental Ethics Review Committee of Shanghai Ocean University.

[0038] 1.2 Main reagents and main instruments

[0039] The main reagents and main instruments are shown in Tables 1 and 2.

[0040] Table 1

[0041]

[0042] Table 2

[0043]

[0044] 1.3 Reagent preparation

[0045] Using dimethyl sulfoxide (DMSO) solution as the solvent, β-sitosterol was dissolved to prepare a stock solution with a concentration of 10 mM. Subsequently, the stock solution was diluted to different concentrations with fresh E3 (NaCl (29.4 g / 100 mL), KCl (1.27 g / 100 mL), CaCl2·2H2O (4.85 g / 100 mL), MgSO4·7H2O (8.13 g / 100 mL)) culture medium, so that the final concentrations of each experimental solution were 10, 50, and 100 μM respectively. Using ddH2O as the solvent, CuSO4·5H2O and LPS were dissolved to prepare stock solutions, and then diluted with fresh E3 culture medium, so that the final concentrations of the experimental solutions were 10 μM and 1 g / L respectively. According to the instructions, DSS was prepared as a DSSR solution with a final concentration of 0.05% using ddH2O as the solvent.

[0046] 2. Experimental methods

[0047] 2.1 Real-time quantitative polymerase chain reaction

[0048] (1) Different concentrations (0, 10, 50, 100 μM) of 1 drug (β-sitosterol) and 3 stimulants (CuSO4·5H2O, LPS, DSS) were used. The subsequent experiments were grouped according to pre-stimulation then drug administration, pre-drug administration then stimulation, simultaneous drug administration and stimulation, with 30 3-dpf zebrafish in each group.

[0049] (2) In the group of pre-stimulation then drug administration, the prepared stimulant was used to treat for 24 h first, then different concentrations of the drug were used to treat for 1 h, and then the larvae were collected and the RNA was extracted and stored at -80 °C.

[0050] (3) In the group of pre-drug administration then stimulation, different concentrations of the drug were used to treat for 1 h first, then the prepared stimulant was used to treat for 24 h, and then the larvae were collected and the RNA was extracted and stored at -80 °C.

[0051] (4) In the group of simultaneous drug administration and stimulation, different concentrations of the drug and the prepared different stimulants were used to treat simultaneously for 24 h respectively, and then the larvae were collected and the RNA was extracted and stored at -80 °C.

[0052] (5) Total RNA was extracted using the Steady Pure Quick RNA Extraction Kit. The concentration of total RNA was measured at A260 / A280 and A260 / A230 nm using a NanoDropTM 1000 spectrophotometer, and the integrity of RNA was detected by 1.5% agarose denaturing gel electrophoresis.

[0053] (6) The required cDNA was obtained by reverse transcribing RNA according to the steps of the reverse transcription kit.

[0054] (7) The standard cycling conditions for real-time quantitative polymerase chain reaction (RT-qPCR) are shown in Table 3.

[0055] Table 3

[0056]

[0057] Results analysis The relative expression levels were calculated using the 2 -ΔΔCt -ΔΔCt method, and the primer sequences are shown in Table 4.

[0058] Table 4

[0059]

[0060]

[0061] 3. Experimental results

[0062] 3.1 Expression changes of inflammasome-related genes in the group with copper ion stimulation followed by drug administration

[0063] Compared with the blank control group, the expression of Drkat7b, Drnek7, and Drcaspase-1 was significantly promoted in the group treated with CuSO4 alone ( Figure 1 A-C). After treatment with β-sitosterol, compared with the group treated with CuSO4 alone, the expression of Drkat7b, Drnek7, and Drcaspase-1 in the group with stimulation followed by drug administration was significantly inhibited. Except for the group treated with 50 μM drug for Drkat7b, in other groups, as the concentration of β-sitosterol increased, the inhibitory effect on the expression of the three genes became stronger.

[0064] 3.2 Expression changes of inflammasome-related genes in the group with drug administration followed by copper ion stimulation

[0065] After pretreatment with β-sitosterol, compared with the group treated with CuSO4 alone, the expression of Drkat7b, Drnek7, and Drcaspase-1 in the group with drug administration followed by stimulation was significantly inhibited ( Figure 2 A-C). The expression level of Drkat7b decreased significantly in the groups treated with 10 μM and 100 μM drugs respectively ( Figure 2 A); the expression level of Drnek7 decreased significantly in the groups treated with different concentrations of drugs, and as the drug concentration increased, the expression level also gradually increased ( Figure 2 B); the expression level of Drcaspase-1 decreased significantly in the groups treated with different concentrations of drugs, and as the drug concentration increased, the expression level gradually decreased ( Figure 2 C).

[0066] 3.3 Expression changes of inflammasome-related genes in the group with copper ion stimulation and drug administration simultaneously

[0067] After the simultaneous treatment with the drug and the stimulant, the expression of three genes was significantly inhibited. Compared with the group treated with CuSO4 alone, after the simultaneous treatment with the drug and the stimulant, the expression of Drkat7b was significantly inhibited at a drug concentration of 100 μM ( Figure 3 A); the expression of Drnek7 was significantly inhibited at different concentrations, and the inhibitory effect was the strongest at a drug concentration of 100 μM ( Figure 3 B); the expression of Drcaspase-1 was significantly inhibited at different concentrations, and the inhibitory effect was the strongest at a drug concentration of 50 μM ( Figure 3 C).

[0068] 3.4 Changes in the expression of inflammasome-related genes after LPS stimulation and drug administration

[0069] Compared with the blank control group, the group treated with LPS alone significantly promoted the expression of Drkat7b and Drcaspase-1, while the expression of Drnek7 did not change significantly ( Figure 4 A, C). After treatment with β-sitosterol, the expression of Drkat7b, Drnek7, and Drcaspase-1 was significantly inhibited. Compared with the group treated with LPS alone, after drug treatment, the expression of Drkat7b was significantly inhibited, and the inhibitory effect was the strongest at a drug concentration of 10 μM ( Figure 4 A); the expression of Drnek7 was significantly inhibited at different concentrations, and the inhibitory effect increased with the increase in drug concentration ( Figure 4 B); the expression of Drcaspase-1 was significantly inhibited after treatment with drugs at different concentrations, and the inhibitory effect was the strongest at a drug concentration of 10 μM ( Figure 4 C).

[0070] 3.5 Changes in the expression of inflammasome-related genes after drug administration followed by LPS stimulation

[0071] After pretreatment with β-sitosterol, the expression of Drkat7b, Drnek7, and Drcaspase-1 was significantly inhibited. Compared with the group treated with LPS alone, after drug treatment, the expression of Drkat7b was significantly inhibited, and the inhibitory effect was the strongest at a drug concentration of 10 μM ( Figure 5 A); the expression of Drnek7 was significantly inhibited after pretreatment with drug concentrations of 10 and 50 μM ( Figure 5 B); the expression of Drcaspase-1 was significantly inhibited, and the inhibitory effect was the strongest at a drug concentration of 10 μM ( Figure 5 C).

[0072] 3.6 Changes in the expression of inflammasome-related genes after simultaneous drug administration and LPS stimulation

[0073] Compared with the blank control group, the expression of Drkat7b and Drcaspase-1 was significantly promoted in the group treated with LPS alone ( Figure 6 A, C). After the drug and LPS were treated simultaneously, the expression of Drkat7b and Drcaspase-1 was significantly inhibited. Compared with the group treated with LPS alone, after the drug and LPS were treated simultaneously, the expression of Drkat7b was significantly inhibited at different drug concentrations ( Figure 6 A); the expression of Drnek7 was significantly increased at the drug concentration of 50 μM ( Figure 6 B), while the expression of Drcaspase-1 was significantly decreased ( Figure 6 C).

[0074] 3.7 Changes in the expression of inflammasome-related genes after administration following DSS stimulation

[0075] After treatment with β-sitosterol, the expression of Drkat7b, Drnek7, and Drcaspase-1 was significantly inhibited. Compared with the group treated with DSS alone, after drug treatment, the expression of Drkat7b was significantly inhibited at the concentrations of 10 and 50 μM ( Figure 7 A); the expression of Drnek7 was significantly inhibited at different drug concentrations, and the inhibitory effect was stronger with the increase of drug concentration ( Figure 7 B); the expression of Drcaspase-1 was significantly inhibited, and the inhibitory effect was also stronger with the increase of drug concentration ( Figure 7 C).

[0076] 3.8 Changes in the expression of inflammasome-related genes after administration first and then stimulation with DSS

[0077] After treatment with β-sitosterol, the expression of Drkat7b, Drnek7, and Drcaspase-1 was significantly inhibited. Compared with the group treated with DSS alone, after drug pretreatment, the expression of Drkat7b was significantly inhibited at different concentrations, and the inhibitory effect was stronger with the increase of drug concentration ( Figure 8 A); the expression of Drnek7 was significantly inhibited at different drug concentrations, and the inhibitory effect was stronger with the increase of drug concentration ( Figure 8 B); the expression of Drcaspase-1 was significantly decreased ( Figure 8 C).

[0078] 3.9 Changes in the expression of inflammasome-related genes after simultaneous administration of drug and DSS stimulation

[0079] After simultaneous treatment with the drug and DSS stimulation, the expressions of Drkat7b, Drnek7, and Drcaspase-1 were significantly inhibited. Compared with the group treated with DSS alone, after simultaneous treatment with the drug and DSS stimulation, the expression of Drkat7b was significantly inhibited at different concentrations, and the expression level of this gene was the lowest when the drug concentration was 50 μM( Figure 9 A); the expression of Drnek7 was significantly inhibited at different drug concentrations, and the expression level of this gene was the lowest when the drug concentration was 100 μM( Figure 9 B); the expression of Drcaspase-1 decreased significantly and decreased with the increase of the drug concentration( Figure 9 C).

[0080] In summary, β-sitosterol can inhibit the inflammation induced by three different stimulants, CuSO4, LPS, and DSS, by inhibiting the expression of inflammasome-related genes in zebrafish, thereby achieving an anti-inflammatory effect. It has broad application prospects for use in the preparation of drugs for treating inflammatory diseases.

[0081] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of β-sitosterol in the preparation of a drug for treating inflammatory diseases, wherein the drug is an inhibitor of inflammasome-related gene expression, and the inflammasome-related gene is selected from one or more of Drkat7b, Drnek7 and Drcaspase-1.

2. The use according to claim 1, characterized in that: The drug uses beta-sitosterol or a pharmaceutically acceptable solvate thereof as an active ingredient, and the use concentration is 0.1 to 100 μM.

3. The use according to claim 1, characterized in that: The medicament further includes a pharmaceutically acceptable carrier or diluent.

4. The use according to claim 1, characterized in that: The dosage form of the drug is selected from at least one of tablets, capsules, granules, pellets, suspensions, syrups, enteric-coated preparations, emulsion suspensions and injections.

5. A pharmaceutical composition for treating inflammatory diseases, characterized in that: The invention comprises β-sitosterol or a pharmaceutically acceptable solvate thereof as an active ingredient, and the concentration thereof is 0.1 to 100 μM.

6. Use of β-sitosterol in the preparation of drugs for treating inflammatory diseases induced by CuSO4, and / or LPS, and / or DSS.

7. The use according to claim 6, characterized in that: The drug is an inflammasome-related gene expression inhibitor, and the inflammasome-related gene is selected from one or more of Drkat7b, Drnek7 and Drcaspase-1.

8. A pharmaceutical composition for treating inflammatory diseases induced by CuSO4, and / or LPS, and / or DSS, characterized in that: The invention comprises β-sitosterol or a pharmaceutically acceptable solvate thereof as an active ingredient, and the concentration thereof is 0.1 to 100 μM.

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